Production of fatty alcohols from engineered microorganisms

Patent metadata

Field	Value
Publication number	US-9650655-B2
Application number	US-201314415665-A
Country	US
Kind code	B2
Filing date	Jul 19, 2013
Priority date	Jul 20, 2012
Publication date	May 16, 2017
Grant date	May 16, 2017

Abstract

Recombinant microorganisms are provided which have been engineered to produce fatty alcohols. Also provided are recombinant microorganisms which comprise a heterologous polynucleotide encoding a fatty alcohol reductase enzyme and an introduced polynucleotide encoding a β-ketoacyl acyl carrier protein synthase.

First claim

The invention claimed is:

1. An engineered prokaryotic cell comprising two recombinant polynucleotides, the first polynucleotide encodes a heterologous fatty alcohol forming reductase (FAR) comprising the amino acid sequence of SEQ ID NO: 8, and the second polynucleotide encodes a β-ketoacyl acyl carrier protein synthase III (FabH).

2. The engineered prokaryotic cell of claim 1 , wherein the engineered prokaryotic cell is a bacterial cell.

3. The engineered prokaryotic cell of claim 2 , wherein the bacterial cell is an E. coli cell.

4. The engineered prokaryotic cell of claim 1 , further comprising a recombinant polynucleotide encoding a thioesterase (TE), wherein the TE comprises an amino acid sequence at least 90% sequence identity to SEQ ID NO: 63.

5. The engineered prokaryotic cell of claim 1 , wherein the TE comprises an amino acid sequence at least 90% sequence identity to SEQ ID NO: 12.

6. The engineered prokaryotic cell of claim 1 , wherein the TE comprises an amino acid sequence at least 95% sequence identity to SEQ ID NO: 12 or SEQ ID NO: 63.

7. The engineered prokaryotic cell of claim 1 , further comprising one or more introduced polynucleotide encoding a FabD and/or FabG enzyme.

8. The engineered prokaryotic cell of claim 7 , wherein said introduced polynucleotide encodes the FabD enzyme.

9. The engineered prokaryotic cell of claim 7 , wherein the FabD enzyme comprises an amino acid sequence at least 90% sequence identity to SEQ ID NO: 26.

10. The engineered prokaryotic cell of claim 7 , wherein said introduced polynucleotide sequence encodes the FabG enzyme.

11. The engineered prokaryotic cell of claim 7 , wherein the FabG enzyme comprises an amino acid sequence at least 90% sequence identity to SEQ ID NO: 18.

12. The engineered prokaryotic cell of claim 7 , wherein the introduced polynucleotide encode the FabD and FabG enzymes, wherein the FabD enzyme has an amino acid sequence at least 90% sequence identity to SEQ ID NO: 26, and wherein the FabG enzyme has an amino acid sequence at least 90% sequence identity to SEQ ID NO: 18.

13. The engineered prokaryotic cell of claim 1 , further comprising an introduced polynucleotide encoding a Fab enzyme selected from a FabI and a FabZ enzyme.

14. The engineered prokaryotic cell of claim 13 , wherein the FabI enzyme has an amino acid sequence at least 90% sequence identity to SEQ ID NO: 20.

15. The engineered prokaryotic cell of claim 13 , wherein the FabZ enzyme has an amino acid sequence at least 90% sequence identity to SEQ ID NO: 22.

16. The engineered prokaryotic cell of claim 13 , wherein the introduced polynucleotide encodes the FabI enzyme and the FabZ enzyme.

17. The engineered prokaryotic cell of claim 1 , further comprising the expression of at least one endogenous genes selected from fadE, fadR, fadD, fabB, fabH and fabF, is attenuated.

18. The engineered prokaryotic cell of claim 1 , further comprising a recombinant acyl-CoA synthase (FadD).

19. A cell culture comprising the engineered prokaryotic cell of claim 1 .

20. A method of producing a fatty alcohol composition comprising
a) providing the engineered prokaryotic cell of claim 1 ;
b) culturing the engineered prokaryotic cell of claim 1 under suitable culture conditions in the presence of a carbon source;
c) producing fatty alcohols; and
d) optionally recovering the fatty alcohols from the engineered cell of claim 1 or from a culture medium thereof.

21. The method of claim 20 , wherein the carbon source comprises a fermentable sugar.

22. The method of claim 21 , wherein the fermentable sugar comprises glucose.

23. The method of claim 21 , wherein the fermentable sugar is obtained from a cellulosic feedstock.

24. The method of claim 23 , wherein the cellulosic feedstock is derived from a biomass selected from grain (e.g. corn), corn stover, corn cobs, wheat straw, bagasse and beet pulp.

25. The method of claim 24 , wherein the biomass has been pretreated.

26. The method of claim 20 , wherein at least 1 g/L of fatty alcohols are produced by the engineered prokaryotic cell.

27. The method of claim 20 , wherein the total fatty alcohol composition produced comprises at least 60% C12 to C14 fatty alcohols.

28. The method of claim 27 , wherein the total fatty alcohol composition produced comprises at least 70% C12 to C14 fatty alcohols.

29. A vector comprising a first polynucleotide encoding a FAR comprising the amino acid sequence of SEQ ID NO: 8, and a second polynucleotide encoding a FabH, wherein the first and second polynucleotides are operably linked to a promoter that is functional in a prokaryotic host cell.

30. A recombinant bacterial culture that produces a composition of fatty alcohols comprising at least 60% C12, C14 and C16 fatty alcohols, wherein the recombinant bacterial culture comprises an engineered bacterial microorganism comprising a gene encoding a heterologous FAR comprising the amino acid sequence of SEQ ID NO: 8 and a gene encoding a heterologous FabH comprising an amino acid sequence at least 90% sequence identity to SEQ ID NO: 10.

31. The recombinant bacterial culture of claim 30 , wherein the engineered bacterial microorganism is an E. coli strain.

32. The recombinant bacterial culture of claim 30 , wherein the fatty alcohol composition produced comprises at least 80% of C12, C14 and C16 fatty alcohols, or comprises at least 60% of the combination of C12 and C14 fatty alcohols.

33. A method of shifting the C12, and C16 fatty alcohols carbon chain length profile in a fatty alcohol composition produced by an engineered bacterial cell comprising a polynucleotide encoding a heterologous FAR having the amino acid sequence of SEQ ID NO: 8, said method comprises introducing a polynucleotide encoding a FabH having at least 90% sequence identity to SEQ ID NO: 10 into the engineered bacterial cell to produce newly engineered bacterial cell, wherein the composition of C12 fatty alcohols produced by the newly engineered bacterial cell has been increased by 25% and the composition of C16 fatty alcohols has been decreased by at least 10% as compared to the engineered bacterial cell.

Assignees

• Codexis Inc

Inventors

• Vroom Jonathan
• Balatskaya Svetlana
• Barak Yoram
• Clark Louis
• Choudhary Trish
• Karlshoej Kristian
• Valle Fernando
• Chan Kaman

Classifications

• C12Y201/01005

  Betaine--homocysteine S-methyltransferase (2.1.1.5) · CPC title

• C12Y301/02027

  Choloyl-CoA hydrolase (3.1.2.27) · CPC title

• C12Y402/01059

  3-Hydroxyacyl-[acyl-carrier-protein] dehydratase (4.2.1.59) · CPC title

• C12Y602/01003

  Long-chain-fatty-acid-CoA ligase (6.2.1.3) · CPC title

• C12N9/88

  Lyases (4.) · CPC title